Epithelial-to-mesenchymal Transition Transition Research Articles

Abstract 465: The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells

Abstract Cancer associated fibroblast (CAF) is a rich source of factors for immune suppression and promotes cancer progression and metastasis via epithelial to mesenchymal transition (EMT). It is well known that tumors with mesenchymal phenotype have significantly poor prognosis and reduced response to checkpoint inhibitors. Targeting CAF becomes an increasingly attractive cancer therapeutic approach. Gremlin-1 is a member of the TGFβ superfamily, expressed by CAFs and tumor cells, and known to play a key role in EMT transition, cancer cell proliferation and stroma maintenance. Gremlin-1 overexpression in CAF or tumor cells often correlates with poor clinical prognosis in multiple cancers including prostate, pancreatic, gastric cancer etc. Here we describe the characterization of TST003 (14E3), a novel humanized IgG1 monoclonal antibody targeting Gremlin-1. TST003 bound to human Gremlin-1 with high affinity and high selectivity, and blocked Gremlin-1 binding to BMP2/4 with an EC50 of 3.68 nM to BMP2 and 4.53 nM to BMP4 respectively. TST003 could dose-dependently reverse Gremlin-1 inhibition of the BMP4-mediated Smad1/5/9 phosphorylation in tumor cells as measured by Western blot. In an ex vivo assay using prostate cancer patient-derived organoids (PDOs), TST003 inhibited the growth of 9 out of 12 human PDOs significantly. TST003 also demonstrated its single agent tumor growth inhibition (TGI>50%) at 10 mg/kg in a human prostate cancer PC3 xenograft model on castrated male nude mice and a human colorectal cancer patient derived xenograft (PDX) mouse model in the presence of human PBMC. Both tumor models expressed Gremlin-1 as detected by IHC analysis. In addition, a significantly better anti-tumor activity was observed when TST003 combined with an anti-VEGFR2 antibody in this CRC PDX (MSS, Kras G12D, PD-L1 negative) model. The safety profiles of TST003 were characterized in the single and repeated dose toxicology studies integrated with safety pharmacology, local tolerance, and in vitro hemolysis study, TCR study, and cytokine release study. TST003 was well tolerated in cynomolgus monkeys without noteworthy abnormalities following a single dose or repeated doses. In summary, TST003 is a first-in-class therapeutic mAb targeting Gremlin-1 overexpressed by CAFs and tumor cells. Our preclinical characterization results provided the rationale for on-going clinical evaluation of TST003 in patients with advanced solid tumors with high unmet medical need either as monotherapy or in combination with SoC. Citation Format: Di Sun, Huanhuan Guo, Chaping Cheng, Hongjun Li, Xinlai Yao, Shuang Lu, Jie Ding, Shenjie Zhang, Shijie Zhou, Steven Yu, Yi Gu, Xueming Qian. The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 465.

Cancer-associated fibroblasts in a 3D engineered tissue model induce tumor-like matrix stiffening and EMT transition.

There is an increasing need for the use of 3D in vitro models over 2D culture. Yet, most models remain unable to fully reproduce the complexity observed in vivo. Tissue-engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture, composition, and cell heterogeneity. Here, we used the self-assembly method and a bladder tissue model to engineer a tumor-like environment, and validated its relevance as a cancer model. The bioengineered bladder cancer model was reconstituted using healthy primary fibroblasts (HFs) and a sub-population of induced cancer-associated fibroblast-like cells (iCAFs) along with a normal urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be more fibrotic with increased collagen remodeling and altered fibronectin content. Moreover, the average stiffness of the ECM scaffolds produced by the iCAFs was 7.4 kPa compared to 1.3 kPa for those produced by HFs. In line with those results, urothelial cells overlaid on the iCAFs-derived ECM was found to be more contractile. We also observed increased nuclear translocation of the mechanosensor YAP1 in urothelial cells overlaid on these iCAFs-derived ECM. Furthermore, urothelial cells seeded on iCAFs-derived stroma displayed invasive behavior compared to urothelial cells on HFs. As increased ECM stiffness and cell contractility are associated with the epithelial-to-mesenchymal transition (EMT), we showed an increase expression of the mesenchymal EMT markers, vimentin and ZEB1, within the urothelium on the iCAFs-derived ECM. Overall, our tissue-engineered tumor model achieved stiffness levels comparable to those observed in bladder cancers along with CAF-mediated ECM remodeling, while the urothelium displayed the biological responses that would be expected within a tumor-like environment. Future experiments will take advantage of the 3D and in vivo-like properties of our engineered model to investigate tumor cell invasion.

Nintedanib-αVβ6 Integrin Ligand Conjugates Reduce TGFβ-Induced EMT in Human Non-Small Cell Lung Cancer.

Growth factors and cytokines released in the lung cancer microenvironment promote an epithelial-to-mesenchymal transition (EMT) that sustains the progression of neoplastic diseases. TGFβ is one of the most powerful inducers of this transition, as it induces overexpression of the fibronectin receptor, αvβ6 integrin, in cancer cells which, in turn, is strongly associated with EMT. Thus, αvβ6 integrin receptors may be exploited as a target for the selective delivery of anti-tumor agents. We introduce three novel synthesized conjugates, in which a selective αvβ6 receptor ligand is linked to nintedanib, a potent kinase inhibitor used to treat advanced adenocarcinoma lung cancer in clinics. The αvβ6 integrin ligand directs nintedanib activity to the target cells of the tumor microenvironment, avoiding the onset of negative side effects in normal cells. We found that the three conjugates inhibit the adhesion of cancer cells to fibronectin in a concentration-dependent manner and that αvβ6-expressing cells internalized the conjugated compounds, thus permitting nintedanib to inhibit 2D and 3D cancer cell growth and suppress the clonogenic ability of the EMT phenotype as well as intervening in other aspects associated with the EMT transition. These results highlight αvβ6 receptors as privileged access points for dual-targeting molecular conjugates engaged in an efficient and precise strategy against non-small cell lung cancer.

Combination Treatment of Withalongolide a Triacetate with Cisplatin Induces Apoptosis by Targeting Translational Initiation, Migration, and Epithelial to Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma.

Treatment regimens for head and neck squamous cell carcinoma (HNSCC) typically include cisplatin and radiotherapy and are limited by toxicities. We have identified naturally derived withalongolide A triacetate (WGA-TA) from Physalis longifolia as a lead compound for targeting HNSCC. We hypothesized that combining WGA-TA with cisplatin may allow for lower, less toxic cisplatin doses. HNSCC cell lines were treated with WGA-TA and cisplatin. After treatment with the drugs, the cell viability was determined by MTS assay. The combination index was calculated using CompuSyn. The expression of proteins involved in the targeting of translational initiation complex, epithelial to mesenchymal transition (EMT), and apoptosis were measured by western blot. Invasion and migration were measured using the Boyden-chamber assay. Treatment of MDA-1986 and UMSCC-22B cell lines with either WGA-TA or cisplatin alone for 72 h resulted in a dose dependent decrease in cell viability. Cisplatin in combination with WGA-TA resulted in significant synergistic cell death starting from 1.25 μM cisplatin. Combination treatment with WGA-TA resulted in lower cisplatin dosing while maintaining the downregulation of translational initiation complex proteins, the induction of apoptosis, and the blockade of migration, invasion, and EMT transition. These results suggest that combining a low concentration of cisplatin with WGA-TA may provide a safer, more effective therapeutic option for HNSCC that warrants translational validation.

EMT status of circulating breast cancer cells and impact of fluidic shear stress

Circulating tumor cells (CTCs) play a vital role in the metastasis and recurrence of breast cancer. CTCs are highly heterogeneous at the stage of Epithelial-to-Mesenchymal Transition (EMT), but the phenotypic and biological characteristics in different EMT stages remain poorly defined. We conducted an orthotopic mouse (4T1) model of breast cancer to isolate CTCs and identified two phenotypes of CTCs: intermediate E/M and mesenchymal CTCs. MTT, Colony formation, Transwell migration and invasion assays were utilized to examined cell proliferation, colony forming, migration and invasion ability. Both the intermediate E/M and mesenchymal CTCs exhibited lower rates of proliferation, colony formation and invasion, as compared to primary tumor cells. The mesenchymal CTCs had a higher rate of invasion but lower rates of proliferation and colony formation than the intermediate E/M CTCs. They also exhibited lower rates of growth and metastasis than the primary tumor cells in vivo, but the mesenchymal CTCs had a higher rate of metastasis than the intermediate E/M CTCs. Fluid shear stress induced the EMT transition of CTCs. The comprehensive analysis of CTCs proteomics discovered proteins that differentially expressed in the two types of CTCs and their primary tumor cells.

Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition.

Simple SummaryThe physical properties of a tumor, such as stiffness, are important drivers of tumor progression. However, current in vitro tumor models fail to recapitulate the full range of physical properties observed in solid tumors. Here, we proposed a 3D self-assembly engineered bladder model using cancer-associated fibroblasts in which stromal cells produce their extracellular matrix. We then proceeded to assess how our model recapitulates biological and mechanical features found in tumors. We confirmed that stroma assembled by cancer-associated fibroblasts have increased extracellular matrix content and display increased remodeling and higher stiffness. Moreover, normal urothelial cells seeded on the tumor model displayed a mechanotransduction response, increased cell proliferation, cell infiltration within stroma, and displayed features of the epithelial-to-mesenchymal transition. Altogether, we demonstrated that our cancer-associated fibroblast-derived tumor stroma recapitulates several biological and physical features expected from a tumor-like environment and, thus, provides the basis for more accurate cancer models.A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium.

Abstract 6230: Loss of ALK4 function promotes cancer progression by regulating cell surface TGF-β receptor stability

Abstract Pancreatic cancer is a lethal disease. More than 50% of pancreatic cancer patients are diagnosed with metastatic disease with a dismal 3% five-year survival rate. There is clearly an urgent need to identify novel targets to treat pancreatic cancer patients. Transforming growth factor-β (TGF-β) signaling is dysregulated during cancer progression, inhibiting tumorigenesis while promoting metastasis. Approximately half of pancreatic cancer patients have mutations of genes in the TGF-β signaling pathway, including SMAD4, SMAD3, TGFBR1, TFGBR2, ACVR1B, and ACVR2A. ALK4 (Activin receptor-like kinase 4), which is encoded by ACVR1B, is a type I receptor in the TGF-β superfamily. Mutation or copy number loss of ALK4 occurs in 34% of pancreatic cancer patients, while ALK4 expression is significantly down-regulated in many cancers, including pancreatic cancer, with low ALK4 expression associated with poor clinical outcomes. Here we demonstrate that loss of ALK4 increases cell migration and invasion, induces epithelial to mesenchymal transition (EMT), and promotes local and distal metastasis in pancreatic cancer models. Loss of ALK4 unexpectedly increases activation of the canonical TGF-β signaling pathway and significantly induces TGF-β-mediated expression of EMT mediators and effectors. Moreover, ALK4 loss increases the cell surface expression of TβRs (TGFβ receptor type I and II), without changing the receptor gene expression, by increasing N-linked glycosylation of TβRs, potentially by altering Golgi morphology and function. Proteomic analysis of newly synthesized proteins induced by ALK4 loss identified galectin-3 (Gal-3) as upregulated. Gal-3 is known to be upregulated by MGAT5 (Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase) and cross-links MGAT5-modified N-glycans on TβRs at the cell surface, suppressing TβRs endocytosis. Consistent with this observation, loss of ALK4 increased MGAT5 mRNA expression and induced Gal-3 protein expression, suggesting that ALK4 might regulate TβRs stability by promoting lattice formation with Gal-3 and preventing internalization. Taken together, our results suggest that the loss of ALK4 increases TβR receptor stability through regulating receptor glycosylation, activating TGF-β signaling, promoting the EMT transition and cancer progression. Citation Format: Manqi Zhang, Jian Chen, Gerard Conrad Blobe. Loss of ALK4 function promotes cancer progression by regulating cell surface TGF-β receptor stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6230.

Abstract 968: Loss of glutathione peroxidase 2 promotes epithelial to mesenchymal transition and breast cancer metastasis

Abstract The processes regulating tumor metastasis are multivariate and complex. Redox regulation of the tumor phenotype by GPx2 knockdown (KD) in breast cancer led us to uncover dramatic effects on spontaneous metastasis. Analysis of single cell RNA sequencing (scRNAseq) data from GPx2 KD tumor and control tumor, revealed that both tumors were comprised of several luminal-like tumor cell clusters and one mesenchymal-like cell cluster (cluster 3). Notably, GPx2 KD promoted a significant increase in the size of mesenchymal cells (cluster 3) relative to control, which might be due to the stimulation of epithelial-to-mesenchymal transition (EMT) in response to GPx2 loss. In support of this view, GPx2 KD stimulated an increase in mRNA expression of basal/mesenchymal (KRT5, KRT14, KRT17, Vimentin, Twist1, Twist2, CDH2) genes and a decrease in mRNA expression of epithelial/luminal (Cldn7 and Epcam) genes, especially in cluster 3. Moreover, GPx2 KD upregulated mRNA expression of basal/mesenchymal (Twist2, CDH2, and KRT14) genes in most luminal-like clusters expressing epithelial/luminal (Epcam, Cldn3/7, CDH1, KRT8/18) genes, implying these clusters may be undergoing EMT transition in a hybrid epithelial/mesenchymal state in response to GPx2 loss. Validation of these data in cell lines and tumors showed that GPx2 KD dramatically enhanced EMT via activation of ROS/HIF1α-mediated signaling. Importantly, these effects were reversed by GPx2 re-expression or HIF1α inhibition, which was capable of suppressing EMT and metastasis. Collectively, these results indicate that GPx2 loss promotes breast cancer metastasis by stimulating EMT due to HIF1α signaling, highlighting the impact of GPx2 and HIF1 on therapeutic intervention in metastasis. Citation Format: Zuen Ren, Huizhi Liang, Malindrie Dharmaratne, Miriam Morales-Gallego, Atefeh Taherian Fard, Jessica Mar, Kimita Suyama, Outhiriaradjou Benard, Michael B. Prystowsky, Larry Norton, Rachel B. Hazan. Loss of glutathione peroxidase 2 promotes epithelial to mesenchymal transition and breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 968.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

The microRNAs miR-302d and miR-93 inhibit TGFB-mediated EMT and VEGFA secretion from ARPE-19 cells

The transforming growth factor-beta (TGFB) plays an essential role in the pathogenesis of some ophthalmologic diseases, including neovascular age-related macular degeneration (nAMD) and proliferative vitreoretinopathy (PVR). TGFB activates the transcription factors SMAD2 and SMAD3 via the TGFB receptor, which together activate several genes, including VEGFA. TGFB treated ARPE-19 cells show an increased proliferation rate and undergo epithelial to mesenchymal transition (EMT). Since microRNAs (miRNAs) are capable of inhibiting the translation of multiple genes, we screened for miRNAs that regulate the TGFB signalling pathways at multiple levels. In this study, we focused on two miRNAs, miR-302d and miR-93, which inhibit TGFB signalling pathway and therefore TGFB-induced EMT transition as well as VEGFA secretion from ARPE-19 cells. Furthermore, we could show that both miRNAs can retransform TGFB-stimulated mesenchymal ARPE-19 cells towards the morphological epithelial-like state. Taken together, transient overexpression of these miRNAs in RPE cells might be a promising approach for further translational strategies.

Capturing the pathway logic of cell signaling for epithelial-mesenchymal transition in ovarian cancer cell lines.

e18063 Background: Epithelial-to- mesenchymal transition (EMT) plays a key role not only in cancer invasiveness and progression but also in treatment resistance. Experimental models suggest that epithelial and mesenchymal phenotypes may represent steady states supported by the regulatory logic of intracellular signaling networks. Using known regulatory interactions between intracellular signaling molecules as a framework, we explored EMT in silico with the aim of identifying potentially novel means of inhibiting metastasis in ovarian cancer. Methods: A mathematical model connecting 27 genes involved in EMT through 153 regulatory interactions was assembled from the Mogrify, Reactome, String and Pathway Studio (PS) databases, the last of which used the MedScan natural language processing (NLP) engine to process over 8,000 full-text publications. Logic model parameters dictating the regulatory response dynamics of the EMT circuit were constrained to values that accurately predicted the proteomic profiles characterized as either epithelial or mesenchymal in 30 cell lines in the publicly available MaxQB database. Results: A total of 94,672 candidate models predicted the protein expression in stable epithelial and mesenchymal cells equally well, clustering into 24 classes of slightly different EMT transition kinetics. Discrete event simulations based on these models identified 7 unique combinations, involving the concurrent modulation of at least 3 gene products capable of effecting this transition successfully even in the presence of noise. Every combination required inactivation of ZEB2, along with 2 other manipulations. Inactivation of TP53, HOXA5, or IRF7 was required in 3 combinations, while inactivation of ICAM1 was required in 2 cases. Only BCL2 was required to be activated in any of these scenarios. Interestingly, BCL2 inhibition is now applied clinically in some hematologic cancers and proving effective in patient-derived cell lines for high-grade serous ovarian cancer when combined with MEK inhibition. Conclusions: This work suggests that EMT might at least in part be driven by normal regulatory interactions between specific proteins in a network of pathways, without the requirement for additional mutations or alterations in the underlying circuitry. Maintenance of ZEB2 or TP53 activation as well as BCL2 inhibition may thus represent promising avenues for future research in arresting EMT and associated chemo-resistance.

Marked epithelial to mesenchymal transition in surgical margins of oral cancer-an in vitro study.

Epithelial to mesenchymal transition (EMT) is a feature of several types of human cancer, including oral squamous cell carcinoma (OSCC). In the present study, tumor and margin cell cultures obtained from patients with OSCC were used to determine the expression patterns of certain EMT-associated markers, including vimentin, α-smooth muscle actin, SLUG and SNAIL. In addition, other EMT-associated features, including clonal, proliferative and migratory potential were compared between the two cell types. Cell cultures were generated from tumor and margin tissue samples from 6 patients and cultured up to the fifth passage. EMT marker expression was assessed by reverse transcription-quantitative PCR. Cell proliferation, colony formation and scratch wound healing assays were conducted to characterize the two cell types in terms of proliferation rates, clonality and motility. All of the studied markers were expressed in tumor and margin cells. Although no significant differences were noted with regard to the aforementioned markers, their expression tended to be higher in margin cultures than in tumor cultures. The expressions of the EMT markers were also higher in the fifth passage compared with those noted at the first with a few exceptions. The rates of proliferation and cell migration were decreased during passages, while the number of colonies was increased in both types of cell culture. Tumor and margin cells indicated certain similarities with regard to EMT transition characteristics.

Inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer\u2013stromal interaction and metastasis

BackgroundExtracellular signal-regulated kinases (ERKs) have been related to multiple cancers, including breast cancer, hepatocellular cancer, lung cancer and colorectal cancer. ERK1/2 inhibitor can suppress growth of KRAS-mutant pancreatic tumors by targeting cancer cell. However, no studies have shown the expression of ERK1/2 on pancreatic stromal and its effect on pancreatic cancer–stromal interaction.MethodsImmunohistochemistry and western blotting were performed to detect the expression of p-ERK1/2 in pancreatic tissues and cells. Cell viability assay was used to study IC50 of ERK inhibitor on pancreatic cancer cells (PCCs) and primary cancer-associated pancreatic stellate cells (PSCs). Transwell migration, invasion, cell viability assay, senescence β-galactosidase staining were performed to determine the effect of ERK inhibitor on PCCs and PSCs in vitro and in vivo. The expression of key factors involved in autophagy and epithelial-to-mesenchymal transition (EMT) process were evaluated by western blotting. The expression of key factors related to cell invasiveness and malignancy were confirmed by qRT-PCR. Co-transplantation of PCC Organoid and PSC using a splenic xenograft mouse model was used to evaluated combined treatment of ERK inhibitor and autophagy inhibitor.ResultsImmunohistochemical staining in pancreatic tumor samples and transgenetic mice detected p-ERK1/2 expression in both cancer cells and stromal cells. In pancreatic tissues, p-ERK1/2 was strongly expressed in cancer-associated PSCs compared with cancer cells and normal PSCs. PSCs were also significantly more sensitive to ERK1/2 inhibitor treatment. Inhibition of ERK1/2 suppressed EMT transition in HMPCCs, upregulated cellular senescence markers, activated autophagy in cancer-associated PSCs; and suppressed cancer–stromal interaction, which enhanced invasiveness and viability of cancer cells. We also found that chloroquine, an autophagy inhibitor, suppressed ERK inhibition-induced autophagy and promoted PSC cellular senescence, leading to significantly decreased cell proliferation. The combination of an ERK inhibitor and autophagy inhibitor suppressed liver metastasis in a splenic pancreatic cancer organoid xenograft mouse model.ConclusionsThese data indicate that inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer–stromal interaction and metastasis.

Abstract 3930: Chemoprevention of metastatic colon cancer by a novel small molecule

Abstract Introduction: Colon cancer is a one of the leading cause of death in both men and women. Metastatic colon cancer is responsible for mortality due to resistance to conventional therapies. Our group has previously reported that activation of AKT and Notch1 plays an important role in metastasis of colon cancer cells. As Notch1 and AKT regulates cell proliferation and are upstream of epithelial to mesenchymal transition (EMT) cascade, it seems interesting to explore their inhibition for pharmacological intervention. In the present study we identified a sesquiterpene molecule, Verrucarin J (VJ), that inhibited Notch1 expression and downregulated AKT mediated EMT transition in colon cancer cells. Methods: The anticancer effect of VJ was assessed on both colon cancer cells and stably AKT overexpressing colon cells by cell proliferation, apoptosis and Western blot analysis. For xenograft studies, pCMV/HCT-116 or AKT/HCT-116 cells (1.5 × 106) in a 50-μl final volume of phosphate-buffered saline (PBS) were injected subcutaneously into separate flanks of the mice. Also effects of VJ were determined in mouse model of colorectal cancer i.e. APC min+/_ mice. Statistical analysis was done by unpaired Student's t-test and one way ANOVA,*p≤0.05, **p≤0.01, ***p≤0.001. Results: Molecular analysis of VJ treatment on colon cancer cells revealed that it inhibited colon cancer growth by down regulating AKT and Notch1 signaling. Western blot analysis revealed that VJ inhibits AKT/NF-κβ/ Bcl2- signaling axis in colon cancer cells. VJ also Inhibited migration and invasion of colon cancer cells that corresponds with downregulation of mesenchymal marker expression. In addition, VJ treatment induces apoptosis in colon cancer cells as well as AKT overexpressing cells. Intraperitoneal administration of VJ in pCMV/HCT116 and AKT/HCT-116 xenograft mice revealed decrease in tumor volume in comparison with control mice. We also examined the in vivo efficacy of VJ in ApcMin/+ mouse model. Treatment of ApcMin/+ with VJ (0.5 mg/kg/IP/twice a week) over 12 weeks significantly reduced the number of intestinal polyps (distal 84%; Middle 63% and Proximal 2%) as compared vehicle treated mice. In addition, we also observed 50% reduction in number of colon tumors in VJ treated mice. Conclusion: In conclusion, our studies suggest that VJ inhibits both AKT and Notch1 signaling and induces apoptosis in colon cancer cells. In vivo studies revealed significant reduction in tumor formation in mice models of colon cancer. Hence VJ could be a viable therapeutic agent for treating patients with metastatic colon cancer. Citation Format: Deeksha Pal, Balaji Chandrasekaran, Ashish Tyagi, Becca Von Baby, Houda Alatassi, Murali Ankem, Chendil Damodaran. Chemoprevention of metastatic colon cancer by a novel small molecule [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3930.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Identification of the tumour transition states occurring during EMT.

In cancer, the epithelial-to-mesenchymal transition (EMT) is associated with tumour stemness, metastasis and resistance to therapy. It has recently been proposed that, rather than being a binary process, EMT occurs through distinct intermediate states. However, there is no direct in vivo evidence for this idea. Here we screen a large panel of cell surface markers in skin and mammary primary tumours, and identify the existence of multiple tumour subpopulations associated with different EMT stages: from epithelial to completely mesenchymal states, passing through intermediate hybrid states. Although all EMT subpopulations presented similar tumour-propagating cell capacity, they displayed differences in cellular plasticity, invasiveness and metastatic potential. Their transcriptional and epigenetic landscapes identify the underlying gene regulatory networks, transcription factors and signalling pathways that control these different EMT transition states. Finally, these tumour subpopulations are localized in different niches that differentially regulate EMT transition states.

A shift from papillary to reticular fibroblasts enables tumour\u2013stroma interaction and invasion

BackgroundTumour stroma consists of a heterogeneous population of fibroblasts and related mesenchymal cells, collectively dubbed cancer-associated fibroblasts (CAFs). These CAFs are key players in cancer invasion of cutaneous squamous cell carcinoma (SCC). As we have shown earlier, papillary and reticular fibroblasts (Pfs and Rfs, respectively) have distinct effects on epidermal and dermal homeostasis, but their role in cancer invasion and epithelial-to-mesenchymal transition (EMT) remains to be determined.MethodsWe used 3D cultures of human skin equivalents (HSEs) to analyse the effects of Pfs and Rfs on the invasive behaviour of SCC and EMT.ResultsWe reveal for the first time the importance of Pfs versus Rfs in SCC invasion and EMT. Cell lines from different stages of SCC showed significantly more extensive invasion into a dermis composed of Rfs than of Pfs. In addition, Rfs-based HSEs showed increased cell activation and stained positive for CAF biomarkers α-SMA and vimentin. Further analysis revealed that invasively growing cancer cells in Rf-HSEs express markers of EMT transition, like SNAIL2, N-cadherin and ZEB1.ConclusionsConversely, our results show that Pfs contain cancer cells more within the epidermis. Rfs are clearly predisposed to differentiate into CAFs upon SCC signals, assisting invasion and EMT.

High expression of hnRNPA1 promotes cell invasion by inducing EMT in gastric cancer

Advanced gastric cancer (GC) has a poor prognosis and its treatment strategies are not very efficient. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has emerged as a plausible GC marker, however the role and molecular mechanism of hnRNPA1 in cell invasion and migration remains unknown. In the present study, the gene expression across normal and tumor tissue (GENT) database was used to evaluate the mRNA expression of hnRNPA1 in various types of cancer. Western blot analysis (WB) and immunohistochemistry (IHC) were performed to detect the protein expression of hnRNPA1 in GC tissues and adjacent non-tumor tissues. The expression of multiple oncogenes was detected by western blot analysis and quantitative RT-PCR in hnRNPA1 overexpressing GC cells. Soft agar colony formation, EdU incorporation, wound healing and invasion assays were applied to verify the role of hnRNPA1 in anchorage-independent cell growth, migration and invasion in GC cells. Epithelial-to-mesenchymal transition (EMT) markers were detected by immunofluorescence, western blot analysis and IHC in vitro. A nude mice model of metastasis carcinoma was established to confirm the role of hnRNPA1 during EMT in vivo. Our results revealed that hnRNPA1 was significantly upregulated in GC tissue. HnRNPA1 overexpression significantly induced cell growth, migration and invasion ability in GC cells. In addition, hnRNPA1 promoted EMT of GC cells in vitro and in vivo. These findings indicated that hnRNPA1 is highly expressed in GC and promoted invasion by inducing EMT transition in GC cells. Thus, hnRNPA1 may be a potential therapeutic target for GC.

Abstract 3794: Selective isolation of epithelial to mesenchymal transitioned circulating tumor cells in NSCLC using novel magnetic nanocubes

Abstract The 5-year survival rate for early to advanced NSCLC patients is 10-5%. Once the tumor metastasizes, the survival rate often drops below 1%. One of the major challenges is to select the patient for appropriate therapy, and the selection process involves invasive biopsy that is difficult to recurrently perform during treatment. Recent studies have shown that liquid biopsy is an attractive alternative. Liquid biopsy involves analysis of either CTCs or ctDNA to understand the tumor therapy. Isolating entire tumor cells provide an opportunity to perform whole genome sequencing for in depth understanding of the tumor. However, frequent changes in cancer signaling and acquired mutations during treatment lead to drug resistance that cannot be diagnosed using current CTC techniques. These oncogenic biochemical modifications that are often associated with metastasis involve upregulation of epithelial to mesenchymal transition (EMT) pathway that change the elasticity of tumor cells for easy shedding into the blood stream. Consequently, EMT transition leads to depletion in epithelial markers such as EpCAM and cytokeratin that are to be targeted. This effect largely limits the use of present technologies utilizing EpCAM as a marker to isolate the CTCs. We hypothesized that these cells can be captured by targeting surface markers that are overexpressed in EM transitioned CTCs. For example, markers such as EGFR and HER2 have been shown to be overexpressed pre and post EMT tumors and therefore can present as a new strategy for non-invasive diagnosis before and during treatment. In this study we have shown that EGFR and HER2 receptors are overexpressed in EGFR and KRAS mutated NSCLC cells. We artificially activated EMT in NSCLC cells and compared the surface biomarker concentrations. Furthermore, we used protein analysis data using western blotting to identify our targets after EMT. Based on the biomarker concentrations, we designed and developed magnetic nanocubes (MNC) surface attached with antibodies to target the selected biomarkers to capture EMT CTCs. The capture efficiency of these magnetic nanocubes was compared in multiple cell lines (HCC827 and A549). Overall we could achieve capture as low as 10 spiked cells in our study. In conclusion, cancer markers such as EGFR and HER-2 that are highly expressed in tumors, once shed as CTCs can be targeted for diagnosis. Therefore, we have developed a novel method to capture EMT CTCs with high selectivity and this method presents a minimally-invasive method for real-time monitoring of patients during drug treatment. Citation Format: Dhananjay Suresh, Shreya Ghoshdastidar, Soumavo Mukherjee, Abilash Gangula, Anandhi Upendran, Raghuraman Kannan. Selective isolation of epithelial to mesenchymal transitioned circulating tumor cells in NSCLC using novel magnetic nanocubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3794. doi:10.1158/1538-7445.AM2017-3794

Molecular Profiling of the Developing Lacrimal Gland Reveals Putative Role of Notch Signaling in Branching Morphogenesis.

PurposeAlthough normal function of the lacrimal gland is essential for vision (and thus for human well-being), the lacrimal gland remains rather poorly understood at a molecular level. The purpose of this study was to identify new genes and signaling cascades involved in lacrimal gland development.MethodsTo identify these genes, we used microarray analysis to compare the gene expression profiles of developing (embryonic) and adult lacrimal glands. Differential data were validated by quantitative RT-PCR, and several corresponding proteins were confirmed by immunohistochemistry and Western blot analysis. To evaluate the role of NOTCH signaling in lacrimal gland (LG) development, we used the NOTCH inhibitor DAPT and conditional Notch1 knockouts.ResultsOur microarray data and an in silico reconstruction of cellular networks revealed significant changes in the expression patterns of genes from the NOTCH, WNT, TGFβ, and Hedgehog pathways, all of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Our study also revealed new putative lacrimal gland stem cell/progenitor markers. We found that inhibiting Notch signaling both increases the average number of lacrimal gland lobules and reduces the size of each lobule.ConclusionsOur findings suggest that NOTCH-, WNT-, TGFβ-, and Hedgehog-regulated EMT transition are critical mechanisms in lacrimal gland development and morphogenesis. Our data also supports the hypothesis that NOTCH signaling regulates branching morphogenesis in the developing lacrimal gland by suppressing cleft formation.